KR20140140080A - Systems and methods for containing biological samples - Google Patents

Abstract

An article for holding a plurality of biological samples comprises a substrate, wherein the substrate comprises a first surface and a second opposite surface and a plurality of reaction sites in the substrate. Each reaction spot extends from an opening at the first surface to an opening at the second surface. The reaction sites include a hexagonal shape and are configured to provide sufficient surface tension by capillary action to retain each biological sample. The reaction site has a density of at least 170 holes per square millimeter over at least a portion of the surface. At least one surface of the surfaces may have a surface roughness characterized by an arithmetic mean roughness (Ra) of 5 nanometers or less.

Description

[0001] SYSTEMS AND METHODS FOR CONTAINING BIOLOGICAL SAMPLES [0002]

The present invention generally relates to devices, systems and methods for containing biological samples, and more particularly to devices, systems and methods for containing biological samples at multiple reaction sites for evaluation.

The use of microtiter plates has been used to monitor / monitor, measure / measure or analyze multiple biological and biochemical reactions during a single experiment or essay. Such plates are often used in sequencing, genotyping, polymerase chain reaction (PCR) and other biochemical reactions to monitor the process and provide quantitative data. For example, an optical excitation beam may be used during a real-time PCR (qPCR) process to illuminate a fluorescent DNA-binding dye or fluorescent probe to produce a fluorescent signal indicative of the amount of the target gene or other nucleotide sequence. The increase in demand to provide more responses per experiment or essay has resulted in the device being able to perform a greater number of reactions simultaneously.

A newer approach, such as digital PCR (dPCR), increases the demand for devices, systems and methods involving a much larger number of sites, much less than those used in more conventional quantitative PCR (qPCR) . There is a need for a system and sample format that provides reliable, high quality data in high density sample formats with sample sites with nanoliter or picoliter or even smaller order volumes .

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention can be better understood from the following detailed description when read in conjunction with the accompanying drawings. These embodiments for illustrative purposes only illustrate novel and unambiguous aspects of the present invention. The drawings include the following drawings:
1 is a top view of a manufactured article according to an embodiment of the present invention.
Figure 2 is a side view of the product shown in Figure 1;
Figure 3 is a cross-sectional view of a portion of the product shown in Figure 1;
Figure 4 is a schematic diagram of an embodiment of the present invention.
5 is a schematic diagram showing results using the model shown in FIG.
Figure 6 shows a pattern for the distribution of response sites according to one embodiment of the present invention.
Figure 7 shows a geometric layout showing a comparison between a circular response spot and a hexagonal response spot.
8 is a cross-sectional view of a substrate according to one embodiment of the present invention.
9 is a perspective view of a portion of a substrate in accordance with an embodiment of the present invention.
10 is a top view of a manufactured article according to an embodiment of the present invention.
Figure 11 is a flow diagram of a method in accordance with an embodiment of the present invention.
12 is a cross-sectional view of a carrier and related substrate in accordance with an embodiment of the present invention.
13 is a top view of the carrier and substrate shown in Fig.
14 is a perspective view of a carrier according to an embodiment of the present invention.
15 is a perspective view of a carrier according to an embodiment of the present invention.
16 is a flow diagram of a method according to an embodiment of the present invention.
17 is a schematic diagram of a system according to an embodiment of the present invention.
18 is a front view of an article of manufacture according to an embodiment of the present invention.
19 to 20 are enlarged front views of the manufactured product shown in Fig.
Figure 21 is a front view of the article of manufacture shown in Figure 18 showing various dimensions of the article.
22 is a front view of an article of manufacture according to an embodiment of the present invention.
23 is a front view of the manufactured product shown in Fig. 22 showing various dimensions of the product.
24 is a flow chart illustrating a method of manufacturing a manufactured article according to an embodiment of the present invention.
25A-25C are cross-sectional views of an embodiment of the product shown in Figs. 18-21 illustrating an embodiment of the method shown in Fig.
26 shows a manufactured article and an associated case or holder according to an embodiment of the present invention.

Embodiments of the present invention generally relate to devices, devices, systems, and methods for monitoring or measuring biological responses to multiple samples or solutions located in a plurality of reaction zones or reaction sites. Examples include the use of polymerase chain reaction (PCR) processes, essays and protocols. It should be appreciated that any suitable PCR method may be used in accordance with the various embodiments described herein if it is generally applicable to dPCR (digital PCR) or qPCR (real-time or quantitative PCR) where multiple samples are processed. Suitable PCR methods include allele-specific PCR, asymmetric PCR, ligation-mediated PCR, multiplex PCR, nested PCR, quantitative or real-time PCR (qPCR), cast PCR, genome walking but are not limited to, genome walking, bridge PCR, digital PCR (dPCR), and the like.

Embodiments of the present invention generally relate to dPCR and qPCR but are not limited to any PCR process, experiment, assay or protocol in which multiple samples or test volumes are processed / processed, observed / observed, measured / The present invention can be applied. In a dPCR assay or experiment according to an embodiment of the present invention, a dilute solution comprising a relatively small number of at least one target polynucleotide or nucleotide sequence is subdivided into a plurality of small test samples or volumes, Some do not contain the target nucleotide sequence. When a sample is subsequently thermocycled in a PCR assay, process, or experiment, an individual sample containing one or more molecules of the target produces an amplified, positive, detectable signal, and the one that does not contain the target (s) Generates a signal that does not generate or is below a predetermined threshold or noise level. Using Poisson statistics, the number of target nucleotide sequences in the original solution can be correlated with the number of samples producing a positive detection signal. In some embodiments, a detected signal can be used to measure the number or number range of target molecules contained in an individual sample or volume. For example, a detection system can be configured to distinguish between a sample comprising one target molecule and a sample comprising two or at least two target molecules. Additionally or alternatively, the detection system can be configured to distinguish between a sample comprising a plurality of target molecules less than a predetermined amount and a sample comprising a predetermined overage amount. In a particular embodiment, a qPCR and dPCR process quantum, essay, or protocol is performed using a single device, device or system.

In various embodiments, the devices, devices, systems and methods described herein may be used to detect an initial sample or one or more types of related biological components or targets contained in a solution. The biological component or target can be a DNA molecule or a DNA molecule comprising a DNA sequence (including a non-cell containing DNA), an RNA sequence, a gene, an oligonucleotide, a molecule, a protein, a biomarker, a cell (for example a circulating tumor cell) (Including, but not limited to) a variety of biological targets. In various embodiments, the biological component may be selected from the group consisting of fetal diagnosis, multiplex dPCR, virus detection, quantitative standards, genetic trait analysis, sequencing assay, experiment or protocol, sequencing verification, mutation detection, detection of genetically modified organisms, May be used in conjunction with one or more PCR methods and systems in areas such as detection and / or copy number variation.

According to an embodiment of the present invention, one or more samples or solutions comprising at least one biological target of interest may be distributed or partitioned between a plurality of small sample volumes or reaction sites. The sample volume or reaction site disclosed herein is generally illustrated as a through-hole between substrate materials; Where applicable, the sample volume or reaction site in accordance with embodiments of the present invention may be a well or indentation formed in the substrate, a spot of solution distributed on the surface of the substrate, or within the test site or volume of the microfluidic system Or may comprise a sample or solution located in or on a small bead or sphere.

In a specific embodiment, the dPCR protocol, assay, process, or experiment involves distributing or splitting the initial sample or solution to at least 10,000 reaction sites, at least 100,000 reaction sites, at least 1,000,000 reaction sites, or at least 10,000,000 reaction sites do. Each reactive site may have a few nanoriters, about one nanoritor or less than one nanorit (e.g., less than 100 picoliters, less than 10 picoliters, and / or less than 1 picoliter) . (E.g., fewer than 1000 target molecules, fewer than 100 target molecules, fewer than 10 target molecules, or only one or two target molecules), the number of target nucleotide sequences contained in the initial sample or solution , It may also be important in certain cases that the total or substantially total content of the initial solution is included or accommodated in the sample volume or reaction site to be treated. For example, if only a few target nucleotides are present in the initial solution, some or all of these target nucleotides are likely to be included in a small residual fluid volume that is not located at any reaction site, It will not be detected, measured or counted. Thus, efficient delivery of the initial solution may reduce the chance or likelihood of miscalculation in the numerical coefficient of the rare allele or the target nucleotide, or, if the target molecule is not successfully located in one of the named reaction sites, It can help reduce the likelihood of not detecting the presence of nucleotides. Thus, embodiments of the present invention can be used to provide high loading efficiency, and high loading efficiency is defined as the volume or volume of the initial sample or solution contained within the reaction site divided by the total volume or mass of the initial sample or solution.

Referring to Figures 1 to 3, in certain embodiments of the present invention, a product, device, substrate, slide or plate 100 includes a plurality of partitions located in the substrate 102, through holes, And includes a base 102 that includes a seat 104. In certain embodiments, the product 100 may comprise a chip. Additionally or alternatively, the article 100 may include a microfluidic device, which may include, for example, a plurality of channels or paths for transporting reagents and / or test solutions to the reaction sites 104 As shown in FIG. In another embodiment, the reaction site 104 comprises a plurality of droplets or beads and the product 100 comprises one or more chambers comprising some or all of the droplets or beads 104 and / Channel. In this embodiment, the droplet or bead 104 can form an emulsion, and some or all of the droplet or bead 104 includes at least one polynucleotide or one or more targets of a nucleotide sequence. If the reaction site 104 is a bead, the beam may optionally include an attached optical signature or label. The droplet or beam 104 may be inspected, monitored, or measured at a time or using one or more droplets or beads 104, for example, using an imaging system, in accordance with an embodiment of the present invention.

In the illustrated embodiment, the article 100 includes a first surface 110 and a second opposing surface 112. In the illustrated embodiment, each reaction site 104 extends from the opening 114 at the first surface 110 to the opening 116 at the second surface 112. While the illustrated embodiment illustrated in Figure 3 represents a substrate comprising through holes 104, substrate 102 may additionally or alternatively comprise other types of reactive sites. For example, the reaction site 104 may be a well or indentation formed in the substrate 102, a spot of solution distributed on the surface 110 or 112, or a reaction volume located in another type of reaction chamber or format, Or a sample or solution located within or on a small bead or sphere.

The reaction site 104 can be configured to provide sufficient surface tension to be pulled by capillary action in each volume of liquid or sample containing the relevant biological component. The article 100 can be any USPN 6,306,578; 7,332,271; 7,604,983; 7,6825,65; 6,387,331; Or 6,893,877, which are incorporated herein by reference as if fully set forth herein. Substrate 102 may be a flat sheet or may comprise any form suitable for a particular application, essay, or experiment. Substrate 102 may comprise any of a variety of materials known in the art including, but not limited to, metals, glass, ceramics, silicon, and the like. Additionally or alternatively, the substrate 102 may comprise polymeric materials such as acrylic, styrene, polyethylene, polycarbonate, and polypropylene materials. The substrate 102 and reaction sites 104 may be formed by one or more of machining, injection molding, hot embossing, laser drilling, photolithography, and the like.

In a particular embodiment, the surfaces 110 and 112 are made of a hydrophobic material, such as described in U.S. Patent Application Publication No. 2006/0057209 or 2006/0105453, which is incorporated herein by reference in its entirety as set forth herein. . ≪ / RTI > In such an embodiment, the reaction site 104 may comprise a hydrophilic material that attracts water or other liquid solution. An array of such hydrophilic zones may include hydrophilic islands on the hydrophobic surface and may be deposited by any suitable technique known in the art, such as deposition, plasma, masking, transfer printing, screen printing, spotting ), ≪ / RTI > or the like, including but not limited to < / RTI >

It has been discovered that a high reaction site density can be configured to reduce the amount of solution remaining on the surfaces 110, 112 during the loading process, resulting in higher loading efficiency or transport of the initial solution. For example, by reducing the ratio of the value of the distance between adjacent wells to the value of the well diameter, the amount of solution left on the surface of the plate can be significantly reduced, so that the initial solution containing the relevant biological component Or all or nearly all of the sample is located within the reaction site 104. In this way, the likelihood of loss of a rare allele or other target molecule is reduced, because it is more likely that one or more target molecules will not remain on the substrate surface instead of being accommodated in one of the designated reaction sites 104 .

Referring to Figure 4, this loading efficiency increase is evidenced by a computer model of a hydrophobic surface comprising a plurality of hydrophilic reaction sites. The model is used to analyze the distribution of samples to multiple reaction sites as a function of the reaction site pitch (or density) for through holes with a diameter of 75 micrometers. Figure 5 shows that a higher percentage of the initial liquid sample is trapped in the reaction site and a smaller amount of residual liquid remains behind the hydrophobic surface after the loading process, while the spacing between reaction sites decreases (density increases). Thus, the higher density of reactive sites 104 of a given cross-sectional dimension provides both an increase in the number of test samples for a substrate 102 of a predetermined size (including the associated rare alleles or other target molecules 0.0 > 110, < / RTI > 112).

In certain embodiments, when reaction sites 104 are imaged by the optical system, there may be a lower limit in the spacing between adjacent reaction sites, for example due to optical constraints. For example, there may be a lower limit in the spacing between adjacent reaction sites due to a limitation of the optical system's ability to clearly image adjacent reaction sites. In order to increase the density of the reaction sites 104 in the substrate 102, a close-packed hexagonal matrix pattern, for example as illustrated in FIGS. 6 and 7, may be used.

The reaction sites having a non-circular cross section advantageously reduce the average distance or spacing between adjacent reaction sites 104 to reduce the amount of residual liquid or solution remaining after the surface 110, 112 after loading of the test solution or sample It was found to be possible. 6 and 7, arrays of hexagonal reaction sites 104 having a vertex-to-vertex diameter D are arranged in a hexagonal pattern, wherein the spacing between adjacent reaction sites or P is the pitch. In a particular embodiment, the cross-talk between adjacent reaction sites in the optical system used to measure the fluorescence signal from the reaction site 104 has a minimum edge distance S between adjacent reaction sites, . Thus, the geometry shown in FIG. 7 can be used and represents the minimum pitch P between reaction sites that still maintains a cross-reaction between adjacent reaction sites below a predetermined value. The dotted circle is also shown in Fig. 7 within each hexagon. It represents the circular reaction site of the diameter D 'having the same value of the edge interval S and the pitch P equal to the hexagonal reaction spot. The gray portion in FIG. 7 represents the area between adjacent reaction sites over a small width W for both circular and hexagonal reaction sites. 7, the area between adjacent reaction sites over the width W is greater in the round reaction site than between the hexagonal reaction sites when the pitch P and the edge spacing S are the same. The modeling results described in connection with Figures 4 and 5 indicate that less area between adjacent reaction sites produces higher loading efficiency. Thus, based on the results shown in FIG. 7, a higher loading efficiency is provided under the same spacing conditions (P and S) for the hexagonal reaction sites than the circular reaction sites.

This result also provides an unexpected advantage over the optical system configured to inspect the reaction site. Since the minimum edge spacing S in Fig. 7 is the same for both circular and hexagonal reaction sites, the cross-reaction between adjacent reaction sites will be the same or similar for any type of reaction site. However, the cross section of the hexagonal reaction spot is larger than the round reaction spot for the same pitch (P) and edge spacing (S). Thus, the image generated by the optical system has a larger area for the hexagonal response spot than the circular response spot. Thus, the larger image produced by the hexagonal response spot can span a potentially large number of pixels. A large number of pixels per reaction spot helps to make a more accurate calculation of the signal produced by the reaction site. Thus, in addition to providing a higher loading efficiency, the use of a hexagonal reaction spot can also provide a more accurate measurement of the optical signal or output produced by each of the reaction sites 104, as shown in Figures 6 and 7, (E.g., measuring or calculating the fluorescence signal generated in proportion to the amount of target or dye molecules).

In the illustrated embodiment shown in FIG. 1, the article 100 has a rectangular shape and an overall dimension of 15 millimeters by 15 millimeters. The article 100 also has a working area, zone or zone 120 with dimensions of 13 millimeters by 13 millimeters. As used herein, the term "area of action", "zone of action" or "zone of action" refers to the surface area, zone or zone of a product (100) containing or distributed with a product, eg, a reaction site or solution volume. In a particular embodiment, the working area of the article 100 may increase to 14 millimeters by 14 millimeters or more, for example, 15 millimeters by 15 millimeters to increase the total number of reaction sites contained in the substrate 102 Lt; / RTI > The article 100 may have different shapes and dimensions. For example, surfaces 110 and 112 may be rectangular, triangular, circular, or some other geometric shape. The total total dimensions and working area 120 of the article 100 may be less or greater than the embodiment shown in FIG. 1, depending on the particular design parameters for a given system, essay, or experiment.

In the illustrated embodiment of FIG. 1, the reaction sites 104 may have a characteristic diameter of 75 micrometers and may be disposed on a working area 120 having a pitch of 125 micrometers between adjacent reaction sites. In another embodiment, the reaction site 104 has a characteristic diameter that is less than or equal to 75 micrometers, for example, less than or equal to 60 micrometers. In another embodiment, the reaction site 104 has a characteristic diameter of less than 20 micrometers, less than 10 micrometers, less than 1 micrometer, or less than 100 nanometers. The pitch between the reaction sites may be less than 125 micrometers, for example less than 100 micrometers, less than 30 micrometers, less than 10 micrometers, or less than 1 micrometer.

In a particular embodiment, the substrate 102 has a thickness between the surface 110 and the surface 112 of about 300 micrometers, and each reaction site 104 has a volume of about 1.3 nanometers. Alternatively, the volume of each reaction site 104 may be less than 1.3 nanometers by, for example, reducing the diameter of the reaction site 104 and / or the thickness of the substrate 102. For example, each reaction site 104 may have a volume less than or equal to 1 nanorit, less than 100, or less than or equal to 10 picoliters. In another embodiment, some or all of the volume of the reaction site 104 is in the range of 1 nanometer to 20 nanometers.

In a particular embodiment, the reaction sites 104 across the surfaces 110, 112 have a density of at least 100 reaction sites per square millimeter. Higher densities are also expected. For example, the density of reaction sites 104 across surfaces 110 and 112 may be at least 150 reaction sites per square millimeter, at least 200 reaction sites per square millimeter, at least 500 reaction sites per square millimeter, More than 1,000 reaction sites, more than 10,000 reaction sites per square millimeter, or more than 1,000,000 reaction sites per square millimeter.

Advantageously, all reaction sites 104 in the working area 120 can be imaged and analyzed simultaneously by the optical system. In a particular embodiment, the working area 120 imaged and analyzed by the optical system includes at least 12,000 reaction sites 104. In another embodiment, the working area 120 imaged and analyzed by the optical system includes at least 15,000, at least 20,000, at least 30,000, at least 100,000, at least 1,000,000 reaction sites, or at least 10,000,000 reaction sites.

In a particular embodiment, the reaction site 104 may have a first plurality of reaction sites, characterized by a first characteristic diameter, a thickness and / or a volume, and a corresponding first characteristic diameter, a second characteristic diameter different from the thickness or volume, And / or a second plurality of reaction sites characterized by volume. For example, such reaction site sizes or variations in dimensions may be used to simultaneously analyze two or more different nucleotide sequences that may have different concentrations. Additionally or alternatively, variations in the size of the reaction site 104 on the single substrate 102 may be used to increase the dynamic range of the dPCR process, the assay, or the experiment. For example, the article 100 may include two or more subarrays of reaction sites 104, each group having a diameter or thickness of the reaction site 104 of the other or remaining group (s) And a different diameter or thickness. Each group can be sized to provide a different dynamic range of the number of counts of the target polynucleotide. The subarrays can be located or located at different portions of the substrate 102 such that the two or more subarrays extend over the entire working area of the product 100 or across a common portion of the working area of the product 100.

In certain embodiments, at least some of the reaction sites 104 are tapered over all or a portion of this wall. For example, referring to FIG. 8, at least some of the reaction sites 104 may include a chamfer 130 at the surface 110. Additionally or alternatively, at least some of the reaction sites 104 may include a chamfer 130 at the surface 112 (not shown). The use of chamfered / chamfered or tapered reaction sites has been found to reduce the average distance or total area between adjacent reaction sites 104 without exceeding the optical limit for solution spacing or minimum spacing between test samples lost. As described above with respect to FIG. 5, a reduction in area between adjacent reaction sites 104 may reduce the amount of liquid solution remaining after surface 110, 112 during the loading process. Thus, a higher sample loading efficiency can be achieved while still maintaining a larger effective spacing between adjacent solution locations or test samples for the optical system.

In the embodiment shown in FIG. 9, the product, device, array, slide or plate 100a includes a non-working area, zone or zone 132a that does not include any reaction sites 104a. The non-working area may be a peripheral zone surrounding the working zone including the reaction site 104a. Alternatively, the non-acting area may comprise an area at the working zone and boundary at one, two or more sides or zones. In the illustrated embodiment shown in Fig. 9, the product 100a has a thickness of 0.3 millimeters or nearly the same, and the distance from the edge of the non-acting area to the working area is 1 millimeter or so; However, other dimensions can be used. In the embodiment illustrated in FIG. 9, the reaction site 104a is 0.075 millimeter or nearly such diameter and 0.100 millimeter, or has almost such a pitch spacing; Other dimensions can be used. Where appropriate, the features and / or dimensions described above with respect to article 100 may be included in article 100a or vice versa.

Referring to Figure 10, in a particular embodiment, a product, device, array, slide or plate 100b includes a functional area, zone or zone 120b and a non-functional area 132b comprising a plurality of reaction sites , The non-working area 132b includes a partition, a divider or a separator 134b located between adjacent working areas 120b. As illustrated in FIG. 10, inactive zone 132b may also include a peripheral zone surrounding working zone 120b. The dimensions shown in Figure 10 for various features of the product 100b are examples of specific embodiments and may vary according to the requirements of a particular design. For example, partition 134b may have a thickness between a working area 120b that is less than 500 microns, less than 1 millimeter, or less than 2 millimeters or 3 millimeters.

The partition 134b can be configured to help isolate the reaction site in one working area, zone or zone from the reaction site in a separate working area, zone or zone. For example, this configuration can be used to facilitate loading of the first sample at the first working area and a different second sample at the second working area, and the two areas are separated by the partition 134b. In a particular embodiment, the working surface 120b and the surface of the partition 134b are flushed to each other on one or both sides of the product 100b. Additionally or alternatively, at least a portion of the partition 134b may be raised or offset from the actuation area 120b on one or both sides of the article 100b. In another embodiment, at least a portion of the partition 134b forms a trough with respect to the working area 120b with respect to one or both sides of the product 100b. If appropriate, the features and / or dimensions described above with respect to article 100, 100a may be included in article 100b, or vice versa.

In a particular embodiment, the substrate 102 comprises a light-shaping material, such as a specific glass or ceramic material. In this embodiment, the method 140 shown in FIG. 11 may be used to fabricate the substrate 102. Advantageously, the last optional member of the method 140 shown in Fig. 11 can be used to provide an opaque or almost opaque substrate 102, so that light emitted from one reaction site 104 is incident on the adjacent reaction site (104).

The method 140 may be used to provide a substrate 102 with sufficient opacity to prevent any or nearly any light emitted from one reaction site 104 from being transmitted from an adjacent reaction site 104 . The method 140 may include removing material from the substrate 102 by an amount sufficient to reduce the thickness between the surfaces 110 and 112, for example by at least 20% relative to the initial thickness or at least 30% Or to remove the material from the substrate 104 by an amount sufficient to reduce the thickness between the surfaces 110, 112 to 40%. The method 140 may also include heating the substrate 102 to a temperature of at least 500 < 0 > C during fabrication. In a particular embodiment, the patterned mask used in method 140 comprises a quartz plate having a chrome pattern. The mask can be removed before exposing at least a portion of the substrate to the corrosive agent. The corrosive material used in method 140 may be hydrofluoric acid.

12 and 13, in a particular embodiment, a first cover 152 having a lower surface 154 and a second cover 156 having a top surface 158 are provided in the carrier 150, The housing 100 is housed. The carrier 150 may further include one or more side walls 159 configured to maintain a predetermined spacing between the covers 152,154. The covers 152 and 154 and the wall 159 together define a cavity 160 sized to include the article 100. During use, the article 100 is disposed within the pores 160 formed between the surfaces 154,158. The thickness of the pupil 160 may be greater than the thickness of the article 100 so that a gap is formed between the article 100 and the lower surface 154 and / exist. As shown in the illustrated embodiment of FIG. 12, there may also be a gap between one or more sidewalls 159. Additionally or alternatively, a portion of product 100 may be attached to one or more covers 152, 156 and one or more sidewalls 159.

The carrier 150 may be made or formed from a metallic material, such as stainless steel, aluminum, copper, silver or gold, or semimetal, such as graphite. Additionally or alternatively, all or a portion of the carrier 150 may be made of a non-metallic material including, but not limited to, glass, acrylic, styrene, polyethylene, polycarbonate, and polypropylene. In a particular embodiment, at least one of the covers 152, 156 includes a material that is optically transparent to provide a window configured to allow optical access to and / or from the reaction site 104. Additionally or alternatively, the entire carrier 150 may be made from one or more transparent or substantially transparent materials.

14, in a particular embodiment, the carrier 150a includes a cover or optical access window 152a that is generally vertically disposed and that can be sized to allow passage of the product 100 to the carrier 150a. An aperture, a port or an aperture 162. The carrier 150a may further include a wiper or blade 164 disposed along at least one long edge of the opening 162. [ The blades 164 may be configured to contact or engage with at least one surface 110, 112 of the article 100 when the article 100 is loaded into the carrier 150a. The carrier 150a is a film or film (not shown) disposed over all or a portion of the opening 162 that penetrates the product 150 when the product 100 is loaded into the carrier 150a, ). ≪ / RTI > In certain embodiments, the membrane and blade 164 form a single piece.

The blades 164 are configured to help distribute the sample fluid to some or all of the reaction sites 104 as the product 100 is inserted into the carrier 150 through the openings 162. In some embodiments, For example, the blades 164 may be configured to contact one or both surfaces 110, 112 during loading of the product 100 such that the liquid does not pass through the blades 164, Is pulled / pulled or pushed by the capillary force into the reaction seat 104 instead of moving past the blade 164. Additionally or alternatively, the blades 164 may be used to remove, for example, liquids, gels, and the like, of the product 100 to reduce or eliminate contamination and / or evaporation of sample fluid contained within the reaction site 104 Or to cover both surfaces 110, 112.

Where appropriate, carrier 150a may include any structure or feature described above with respect to carrier 150, or vice versa.

15, in a particular embodiment, carrier 150b includes a body 170 that may include some or all of the structure and features of carrier 150 and / or carrier 150a. The carrier 150b may be a loader or insertion tool for retaining the product 100 for assisting in / helping to load the product 100 into the body 170, Lt; RTI ID = 0.0 > 172 < / RTI > The tool 172 may have a U-shaped body, and the product 100 is secured within the U 'prior to loading into the body 170. The tool 172 may include a tab 174 on an opposite arm 175 configured to be fastened or compressed to a corresponding tab or similar structure 176 of the body 170.

The portion of the pores 160 between the product 100 and the surfaces 154 and 158 prevents evaporation of the test solution contained from the reaction site 104 without mixing with the test solution contained in the reaction site 104 May be filled with an immiscible fluid 170 (e. G., A liquid or gel material) configured to reduce the volume of the fluid. One fluid 170 suitable for a variety of applications is Fluorinert, commercially available from 3M Company. However, in certain embodiments, fluarinuts may be a problem in certain PCR applications due to its tendency to form an unwanted air bubble that easily fills the air that can later be released during PCR cycling.

Alternatively, it has been found that, in certain embodiments, polydimethylsiloxane (PDMS) can be used in the pores 160 when the PDMS is not sufficiently cross-linked. In these embodiments, PDMS has been found to have several features that make it suitable for use with PCR, including low spontaneous fluorescence, thermal stability at PCR temperatures, and non-inhibition of polymerization processes. In addition, the PDMS may comprise an aqueous sample, but it may be a gas which is permeable to water vapor. Typical siloxanes for cross-linking agents used in general applications, but not in embodiments of the present invention, are 10: 1 by weight (10% cross-linking agent).

By under-cross-linking the PDMS material, the resulting material can act as a suitable encapsulant to reduce evaporation while also retaining the good properties associated with the fully crosslinked material described above Was found. More specifically, an under-crosslinked PDMS material can be formed by using less than 10% by weight of a cross-linking agent. For example, crosslinking levels of up to 1% by weight have been found to meet design requirements for particular PCR fields, such as certain dPCR domains. A number of dPCR reactions have been demonstrated using an encapsulated plate (100) with the amount of cross-linking agent being 0.8 wt% or less. In addition, due to the higher viscosity of the under-crosslinked PDMS material compared to fluarinuts, the PDMS encapsulant can also provide packaging requirements and a consumer workflow solution on its own .

Referring to FIG. 16, a method 200 for producing a plurality of biological samples includes providing a description of an article, e.g., article 100, 100a or 100b. Method 200 further includes providing a carrier, e.g., carrier 150, 150a or 150b, and providing an insertion tool, e.g., insertion tool 172, wherein the insertion tool is configured to slideably fasten And a U-shaped body including a pair of arms. The method 200 also includes mounting or attaching the substrate to the insertion tool and mounting or attaching the insertion tool to the carrier. In certain embodiments, the substrate is mounted or attached to the insertion tool, and then the insertion tool and substrate are mounted together on the carrier. In another embodiment, the insertion tool is mounted on a carrier without a substrate, and the substrate is subsequently mounted on the insertion tool and / or carrier.

Once the substrate is mounted or attached, the method 200 may include sliding the insertion tool along the carrier by an amount sufficient to position the substrate within the carrier, for example, by inserting the substrate through the opening and / . Using the method 200, the solution or sample can be applied to the side of the substrate in such a way that the solution is deposited or drawn through the reaction site or through hole in the substrate as the substrate is inserted into the carrier. In addition, one of both surfaces of the substrate may be covered with a liquid or gel to protect the solution, for example from contaminants and / or evaporation.

In certain embodiments, at least 99% of the liquid sample is received by at least some of the reaction sites. In another embodiment, at least 99.5% or 99.9% of the liquid sample is accommodated by at least some of the reaction sites. In a particular embodiment, the total volume of reaction sites 104 is selected to be greater than the volume of the liquid sample loaded into the reaction site 104. This has been found to increase the loading efficiency which may be important in certain situations as described above. In a particular embodiment, the ratio of liquid volume sample to total volume of all reaction sites 104 is 95% or less. In another embodiment, the ratio of liquid volume sample to total volume of all reaction sites 104 is less than 90%, less than 80%, or less than 70%. In a particular embodiment, the value of this analogy depends on the percentage of the total volume of each reaction site that is filled with liquid after loading. For example, if only 90% of each reaction site 104 comprises a liquid sample after loading, the ratio of the liquid volume sample to the total volume of all reaction sites 104 is 90% or less, 80% or less, 70% or less Or less or 60% or less.

Various methods and devices can be used to provide detection of one or more relevant biological components contained in the reaction site 104. For example, the various fluorescent dyes may be incorporated into a solution comprising one or more biological components involved, and then the optical system may be used to detect this component to detect the presence or amount of one or more biological components. In another embodiment, the presence of an ion (cation or anion) can be detected and / or changes in pH, voltage or current can be used to detect the presence or amount of one or more biological components involved.

Referring to FIG. 17, a system 400 may be used to optically view, inspect, or measure one or more samples or solutions containing related biological components contained in the reaction site 104 of the article 100. The article 100 may be included in a carrier, e.g., carrier 150, 150a or 150b. The system 400 includes an optical bead or system 402. The system 400 further includes a controller, computer or processor 404 configured to manipulate various components of, for example, the optical system 402 or to acquire / acquire / process data provided by the system 400. For example, the computer 404 may be used to obtain / obtain / process optical data provided by one or more light detectors of the optical system 402. In certain embodiments, processor 404 may send data to one or more computing systems for further processing. Data may be transmitted from the processor 404 to the computing system via an Internet connection or some other network system.

In a particular embodiment, the system 400 includes a thermal control system 406 including, for example, a thermal cycler configured to perform a PCR procedure or protocol on at least some of the samples included in the product 100 . The systems 402 and 406 may be coupled or coupled together in a single unit to perform, for example, qPCR and / or dPCR procedures, essays, experiments, or protocols in at least some of the samples included in the product 100 . In this embodiment, the computer 404 may be used to control and / or control the systems 402 and 406, or may collect or process data provided or obtained by the systems 402 and 406. Alternatively, thermal control system 406 may be completely separate from optical system 402 and / or computer 404. In this embodiment, after performing a thermal cycle in the sample using the thermal control system 406 or some other thermal controller or thermal cycler, the optical system 402 is used to determine the temperature of the sample contained in the reaction spot 104 dPCR or end-point PCR procedures. In certain embodiments, the thermal control system 406 performs PCR using conventional thermal cycler, isothermal amplification, thermal convention, infrared mediated thermal cycling, or helicase dependent amplification Lt; / RTI > In a particular embodiment, at least a portion of the thermal control system 406 may be integrated into the product 100 or into the product 100. For example, the article 100 may include one or more heating elements distributed along one or both surfaces 110, 112. Additionally or alternatively, at least a portion of the substrate 102 may be made of a material having an electrical resistance configured to provide resistive heating, for example during application of a voltage potential to the substrate 102, have.

In a particular embodiment, the article 100 includes electronic chips including integrated circuits and semiconductors. In this embodiment, the detection system can also be integrated into the chip to measure the presence and / or amount of the relevant biological components.

The optical system 402 includes a light source 410 and an associated excitation optical system 412 configured to illuminate at least some of the sample contained in the reaction spot of the article 100. In some embodiments, The excitation optical system 412 may include one or more lenses 414 and / or one or more filters 416 for conditioning the light directed to the sample. The optical system 402 may further include a light detector 420 and associated emission optical system 422 configured to receive optical data emitted by at least some of the samples contained in the reaction site of the article 100 . For example, when the system 400 is configured to perform a qPCR and / or dPCR essay or experiment, the sample may provide a fluorescent signal that varies depending on the amount of target nucleotide sequence contained in the various response sites of the product 100 Fluorescent dyes. The emission optical system 422 may include one or more lenses 424 and / or one or more filters 426 for conditioning the light directed to the sample.

In the illustrated embodiment of FIG. 17, excitation / emission optical systems 412 and 422 both include one or more common optical members. For example, the excitation / emission optical systems 412 and 422 both include beamsplitters 430 that reflect the excitation light and transmit the emitted light from the sample to the optical detector 420. In certain embodiments, the excitation / emission optical systems 412 and 422 can both be used to improve optical performance to provide more uniform illumination and reading of light into and out of the sample included in, for example, And an objective lens (not shown) disposed between the beam splitter 430 and the product 100, In certain embodiments, for example, if equal illumination is less important (e.g., in some dPCR fields), the common objective can be omitted, as shown in the illustrated embodiment of FIG. Omission of the objective lens can help to reduce the size and complexity of the optical system 402.

The light detector 420 may include one or more photodiodes, a photomultiplier tube (PMT), and the like. For example, such optical detectors may be used when the optical system 402 is configured to scan a separate set of reaction sites 104 or a subset of reaction sites 104. In another embodiment, the light detector 420 may include one or a fragmented detector array, for example, one or more CCD (charge coupled device) or CMOS (complementary metal-oxide semiconductor) arrays. A fragment detector array may be advantageously used when all or most of the groups of reaction sites 104 are imaged or inspected simultaneously. To provide a plurality of pixels for each reaction spot, the light detector 420 may include at least 4,000,000 pixels or more than 10,000,000 pixels.

In a particular embodiment, the article 100 includes electronic chips including integrated circuits and semiconductors. In such an embodiment, the detection system can also be integrated into the chip to measure the presence and / or quantity of the biological components involved.

Referring to Figures 18-21, in certain embodiments, a product, device, array, slide, or plate 500 includes a reaction site 504 located on a substrate 502 or substrate 102 comprising a plurality of through- . The substrate 502 includes a first surface and a second opposing surface. In the illustrated embodiment, each reaction spot 504 extends from the opening at the first surface to the opening at the second surface. As shown in FIGS. 19 and 20, the reaction sites 504 may have a hexagonal shape and / or may be arranged in a dense fill hexagonal matrix pattern. Alternatively, some or all of the reaction sites 504 may have the shape, diameter, density, thickness, pitch spacing, etc. described above in connection with the reaction site 104. The article 500 further includes one or more tabbed cutout or blank zones 506 in which no reaction sites 504 are present. The blank zone 506 may be located in a support zone for the article 500, as described below. In the illustrated embodiment, the blank zone defines four semicircular features; Other shapes and sizes are expected. In addition, the article 500 may include a blank perimeter 508 in which no reaction sites are located.

In certain embodiments, the substrate 502 includes silicon that can be configured to provide an even temperature distribution across the product 500 during use. Alternatively, the substrate 502 includes a glass material, such as an optical structured glass ceramic or metal, such as aluminum, copper or stainless steel.

Referring to FIG. 19, the reaction sites 504 may be arranged to define one or more dropout zones 509 located within the array of reaction sites 504. In some embodiments, the drop zone 509 has dimensions (e.g., visible to the naked eye, without the use of a magnifying device) that are viewable to the naked eye. In the illustrated embodiment, product 500 includes one dropout zone 504 located in the first quadrant of product 500; Multiple dropout zones on a single product 500 may be included. One or more dropout zones 509 may define a longer overall shape along one axis rather than an orthogonal axis as shown in FIG. Thus, the use of a single elongated dropout zone 509 shown in Fig. 19 located far from the center of the product 500 (e.g., determining which side is front and rear, To determine the orientation of the product 500 (to determine the proper orientation for the axis perpendicular to the page of Figure 20). The dropout zone 509 may also be configured to provide a reference signal, e.g., a reference optical signal, that is used during the optical inspection of the reaction spot 504.

In a particular embodiment, the plurality of dropout zones 509 may include, for example, a dropout shape (s), a number of dropout zones 509, and / or one dropout zone 509 for the other dropout zones 509 509 based on the relative position of the product 500. [ For example, the number of dropout zones in a particular product 500 may be used to measure the diameter of the reaction sites 504 and / or the distance between the dropout zones 509, The geometry of the substrate 509 can be used to measure the number of reaction sites 504 or the pitch between the reaction sites 504. Many different combinations of dropout zone size, shape, and distribution are expected.

Referring to Fig. 21, the product 500 may have a total dimension of about 10 mm x 10 mm. Fig. 21 also shows other dimension values associated with the particular embodiment shown in Fig.

Referring to Figures 22 and 23, the product 500b can also include one or more landing zones 530 that can be sized to provide better loading characteristics, May be configured similar to product 500 in Fig. Thus, one or more edges of product 500b have a wider zone without reactive sites 504 than the other edges of product 500b.

The product 500 may, if appropriate, integrate various members and / or features described in connection with the product 100, or vice versa. Also, according to embodiments of the present invention, product 500 may be used in carrier 150 or other carrier. Product 100 may use product 500 in conjunction with system 400 or method 140 and in conjunction with other systems and methods disclosed herein in connection with product 100 in a manner similar to that described herein.

In certain embodiments, the substrate 502 comprises a silicon material and the reaction sites 504 may comprise through holes formed using a hexamethyldisilazane (HMDS) vapor coating process. Referring to FIG. 24, a method 600 may be used to form the through hole reaction sites 504 in the silicon substrate 502. The method 600 includes a process 605 that includes applying or forming a pattern at a first surface of a substrate material. The method 600 further includes a process 610 comprising etching a plurality of wells at a first surface of the substrate using a pattern. The method 600 also includes a process 615 that includes removing material from a second surface opposite the first surface. The method 600 further includes a process 620 that includes etching and / or polishing the second surface. The method 600 further includes a process 625 that includes coating at least one surface to form, for example, a hydrophobic surface.

Referring to FIGS. 25A-25C, method 600 may include a deep reactive-ion etching (DRIE) process to form reaction sites 504 in product 500. As shown in FIG. 25A, the article 500 includes a first surface 510 and a second opposing surface 512. 25A shows a mask 550 applied to a second surface 512 according to a method 600. Using an etch process such as DRIE, the mask 550 may be configured to form a plurality of wells 504 'at the second surface 512. For purposes of illustration, product 500 is shown in a horizontal orientation, while second surface 512 is located below first surface 510; It is understood that during fabrication and / or use, the second surface 512 may be disposed on the first surface 510 and / or the product 500 may have a different orientation, e.g., a vertical orientation. 25B, the well 504 'does not penetrate the first surface 510, but has a depth less than the thickness of the product 500. As shown in FIG. Alternatively, the etching process 610 may produce a through hole that completely penetrates the thickness of the product 500. In this embodiment, additional processing of the first surface 510 may or may not be performed in accordance with the method 500.

Referring to FIG. 25C, a first surface 510 (FIG. 25) is formed in accordance with process 615 of method 600 to reduce the thickness of product 500 by an amount sufficient to form through hole 504 from well 504 ' ) Can be further processed. According to process 620 and / or 625 of method 600 to produce first surface 510 such that the sample or solution applied to first surface 510 is effectively received by through hole 504, Lt; RTI ID = 0.0 > 512 < / RTI > Additionally or alternatively, process 620 and / or 625 may be performed on second surface 512. In this embodiment, the etch process 615 may be omitted or performed altogether so that the final shape of the product 500 is a substrate having a plurality of wells 504 'instead of the through holes 504 shown in Figure 25C .

In a particular embodiment, the process 620 and / or 625 is performed such that the at least one surface 510, 512 has a lower roughness than the predetermined value. For example, after the solution or sample is introduced into the reaction site 504, the remaining thin film of solution or sample may be left behind (e.g., during the PCR thermal cycling process) or may be subsequently formed at the first surface 510 . This residual film may provide a " bridge " between neighboring reaction sites 504. The bridging layer may contaminate one reaction site 504 by one or more adjacent or neighboring reaction sites 504. To solve this problem, it has been found that this bridging problem can be solved or eliminated when the first surface 510 is polished and / or coated to have a lower roughness than the predetermined value. For example, when the product 500 includes the silicon material and the reactive geometry shown in FIG. 20, if the roughness of the first surface 510 satisfies any following roughness criteria, then the bridging problem is eliminated It was decided to:

Ra (arithmetic mean): 5 nm or less.

ㆍ Rv (maximum valley depth): 15 nm or less.

ㆍ Rp (maximum peak height): 9 nanometers or less.

Rt (maximum peak to trench): 24 nanometers or less.

In a particular embodiment, the article 100 or 500 may be used for any enclosure, housing or case disclosed in co-pending U.S. Provisional Application No. 61 / 723,710, the contents of which are incorporated herein by reference Lt; / RTI > For example, as shown in FIG. 26, the article 500 may be arranged in an enclosure, a housing, or a case 700 according to an embodiment of the present invention and provisional application 61 / 723,710. The case 700 may include a base 702 and a cover or lid 704 configured to be sealably fastened to the base 702. Base 702 and cover 704 may be joined together to form a cavity or chamber 708 that can receive and contain product 500. The article 500 may be part of the base 702, separated from the base 702, separated or separated, and configured to be mounted and fixed by the base 702.

The base 702 includes a plurality of bosses, taps, staking seats, or support pads 720 (not shown) configured to secure / fix or position the product 500 within the base 702 and the pores 708 For example tabs 720a and 720b in the illustrated embodiment). One or more tabs 182 may be staked to deform or move the material from the tabs to firmly secure the product 500 within the base 702. Additionally or alternatively, the article 500 can be glued to one or more tabs 182 using an adhesive, an epoxy, or a tackifier. In a particular embodiment, a glass or silicone product 500 (e.g., glass) is used to avoid possible cracking or damage to such a holder material induced by the use of crimping or hiding of the force generated by the tab 720. [ ). ≪ / RTI > In the illustrated embodiment, the tab 720a conforms to the blank area 506 of the product 500. The tab 620a and the blank region 506 are sufficiently large to provide adequate support of the product 500 but the effective area of the corresponding product 500 is less than the predetermined number of reaction sites 504, Lt; RTI ID = 0.0 > predetermined < / RTI >

The foregoing presents a description of the best mode contemplated for carrying out the invention, how it is made and used, and the manner in which those of ordinary skill in the art, having the benefit of the full and clear, concise and exact, . However, it is to be understood that the present invention may be embodied in other specific forms that are fully embodied and construed in alternative and alternative forms from those described above. As a result, there is no intention to limit the invention to the specific embodiments disclosed. On the contrary, the intention is to cover variants and alternatives falling within the spirit and scope of the invention as generally represented by the following claims, which specifically state and describe the subject matter of the present invention.

Exemplary systems for methods related to the various embodiments described in this document include those described in the following US Provisional Application:

U.S. Provisional Application No. 61 / 612,087, filed March 16, 2012; And

U.S. Provisional Application No. 61 / 723,759, filed November 7, 2012; And

U.S. Provisional Application No. 61 / 612,005, filed March 16, 2012; And

U.S. Provisional Application No. 61 / 612,008, filed March 16, 2012; And

U.S. Provisional Application No. 61 / 723,658, filed November 7, 2012; And

U.S. Provisional Application No. 61 / 723,738, filed November 7, 2012; And

U.S. Provisional Application No. 61 / 659,029, filed June 13, 2012; And

U.S. Provisional Application No. 61 / 723,710, filed November 7, 2012; And

U.S. Provisional Application No. 61 / 774,499, filed March 7, 2012; And

Life Technologies, LT00656 PCT, filed March 15, 2013; And

Life Technologies, LT00657 PCT, filed March 15, 2013; And

Life Technologies, LT00658 PCT, filed March 15, 2013; And

Life Technologies, LT00668 PCT, filed March 15, 2013; And

Life Technologies, Inc. filed on March 15, 2013, LT00699 PCT.

All of these applications are also incorporated herein by reference in their entirety.

Claims (63)

1. An article having a plurality of biological samples,
A substrate comprising a first surface and a second opposing surface; And
Wherein each of the reaction sites extend from an opening at the first surface to an opening at the second surface and the reaction sites are capillary to retain respective biological samples, Lt; RTI ID = 0.0 > a < / RTI >
Wherein the density of reactive sites on at least a portion of one of the surfaces is at least 170 holes per square millimeter. At least one of the surfaces has an arithmetic mean roughness (Ra) of less than or equal to 5 nanometers, a maximum valley depth illuminance (Rv) of less than or equal to 15 nanometers, a maximum peak height illuminance (Rp) of less than or equal to 9 nanometers, A surface roughness characterized by at least one of a peak roughness (Rt) for a maximum trench less than or equal to 24 nanometers. The article of claim 1, wherein at least two of the reaction sites include a non-circular shape at one of the surfaces or at each of the surfaces. The article of claim 1, wherein at least two of the reaction sites include a hexagonal shape at one of the surfaces or at each of the surfaces. 2. The method of claim 1, wherein the reaction site is selected from the group consisting of a through hole, a well, an indentation, a chemically modified spot at one of the surfaces, a hydrophilic spot at the other hydrophobic surface product. The article of claim 1, wherein the substrate comprises a flat plate. The article of claim 1, wherein each of the opposing surfaces comprises a hydrophobic material, and wherein at least two of the reaction sites include a hydrophilic material. The article of claim 1, wherein the openings in each surface are arranged in a tightly packed hexagonal matrix. The article of claim 1, wherein the substrate has a thickness of 300 micrometers or less. The article of claim 1, wherein each of the plurality of reaction sites has a diameter of 60 micrometers or less. 2. The apparatus of claim 1, wherein the reaction spot diameter of the first plurality of reaction sites is different from the reaction spot diameter of the second plurality of reaction sites. The article of claim 1, wherein each of the walls of the two or more reaction sites is tapered between the surfaces. 13. The article of claim 12, wherein the taper is greater than 3 degrees. 2. The article of claim 1, wherein each of the two of the at least two reaction sites comprises a chamfer located at one of the surfaces or at each of the surfaces. The article of claim 1, wherein at least some of the reaction sites have a volume between the opposing surfaces of less than 10 nanometers. The article of claim 1, wherein at least some of the reaction sites have a volume between the opposing surfaces of less than or equal to 1 nanoritre. The article of claim 1, wherein at least some of the reaction sites have a volume between the opposing surfaces of less than 100 picoliters. The article of claim 1, wherein at least some of the reaction sites have a volume between the opposing surfaces of less than 30 picoliters. 2. The apparatus of claim 1 wherein the reaction site volume of the first plurality of reaction sites is different from the reaction site volume of the second plurality of reaction sites. The article of claim 1, wherein the density of reaction sites is at least 170 holes per square millimeter. The article of claim 1, wherein the density of reaction sites is at least 200 holes per square millimeter. 2. The article of claim 1, wherein the minimum spacing between reaction sites adjacent to the plurality of reaction sites is less than 85 micrometers. The method of claim 1, wherein the surfaces of the substrate have a dimension of at least 14 millimeters by 14 millimeters, the surfaces defining a working area comprising the plurality of reaction sites, the working area being at least about 13 millimeters by 13 millimeters Products. The article of claim 1, wherein the substrate comprises at least 20,000 reaction sites. The article of claim 1, wherein the substrate comprises at least 30,000 reaction sites. The article of claim 1, wherein the substrate comprises at least 100,000 reaction sites. The article of claim 1, wherein the substrate comprises at least 1,000,000 reaction sites. The article of claim 1, wherein the substrate comprises an optically structured glass ceramic material. The article of claim 1, wherein the substrate comprises a silicone material. The apparatus of claim 1, further comprising a fiducial disposed on at least one of the surfaces. 1. An article for retaining biological samples for analysis, said article comprising:
A substrate having a pair of opposing surfaces; And
Wherein each of the reaction sites extends from an opening at one of the opposite surfaces of the substrate to an opening at the other of the opposite surfaces, To provide sufficient surface tension by capillary action to retain biological samples;
Wherein at least some of said reaction sites have a volume between said opposing surfaces of less than or equal to 1 nanoritre. The article of claim 1; And
An apparatus comprising a carrier, the carrier comprising:
A first cover including a lower surface; And
And a second cover including an upper surface,
Wherein the article is disposed between the upper surface and the lower surface;
Wherein there is a space between the first surface of the substrate and the upper surface;
Wherein there is a space between the second surface of the substrate and the lower surface. 33. The apparatus of claim 32, further comprising a gel material disposed between the spaces. 33. The apparatus of claim 32, further comprising a PDMS material disposed within the spaces. 33. The article of claim 32, wherein at least one of the covers comprises a window configured to provide optical access to the reaction sites. 33. The article of claim 32, wherein the carrier comprises an aperture sized perpendicularly to the surfaces and sized to allow passage of the substrate. 37. The article of claim 36, wherein the aperture comprises a penetrating membrane that breaks when the substrate is moved into the carrier. 33. The method of claim 32,
A U-shaped body configured to secure the product for loading into the carrier; And
Further comprising an insertion tool including a pair of arms configured to slideably engage with the carrier;
Wherein the insertion tool is configured to carry the product to the carrier. 41. The apparatus of claim 38, wherein the carrier comprises a wiper blade configured to contact at least one of the surfaces of the article while loading the article onto the carrier. 32. The apparatus of claim 32,
A light source configured to provide an excitation beam;
A photodetector configured to receive emitted light from the at least two of the reaction sites when the excitation beam interacts with a biological solution contained in at least two of the reaction sites; And
An excitation optical system configured to transmit at least a portion of the excitation beam to the at least a portion of the reaction sites, and an excitation optical system configured to transmit at least a portion of the emitted light from the at least two reaction sites to the detector. ≪ / RTI > 41. The system of claim 40, further comprising a thermal conditioning unit for controlling the temperature of the substrate or within the substrate. 41. The method of claim 40,
Control of the light source;
Control of the photodetector;
Control of an optical member of the optical system; or
Controlling the temperature of at least one of the solution or the solution contained within at least one of the reaction sites; or
Further comprising a microprocessor configured to provide at least one of processing of data obtained by the light detector. 41. The article of claim 40, wherein the light detector comprises a CCD array or a CMOS array. 43. The article of claim 43, wherein the light detector comprises at least 4,000,000 pixels. 43. The article of claim 43, wherein the light detector comprises at least 10,000,000 pixels. CLAIMS What is claimed is: 1. A method of producing a product having biological samples for analysis,
Providing a substrate comprising glass and having a first surface and a second opposing surface;
Covering the glass with a mask comprising a pattern;
Passing an ultraviolet beam through at least a portion of said substrate through said mask;
Exposing at least a portion of the substrate to a caustic agent;
Removing material to provide a plurality of reaction sites extending from one of the surfaces of the substrate to the other of the surfaces; And
And subsequently exposing the substrate to an ultraviolet beam to alter the characteristics of the glass. 47. The method of claim 46, wherein the glass is a stereolithographic material. 47. The method of claim 46, wherein said characteristic is opacity of said glass, said modification being such that light emitted from one reaction site of said plurality of reaction sites is carried through said glass to a reaction site adjacent said plurality of reaction sites Wherein the opacity of the glass is increased by an amount sufficient to prevent the glass. 47. The method of claim 46, further comprising removing material from the substrate by an amount sufficient to reduce the thickness between the surfaces of the substrate. 47. The method of claim 46, further comprising heating the substrate having the reaction sites to a temperature of at least 500 < 0 > C. 47. The method of claim 46, further comprising removing material from the substrate by an amount sufficient to reduce at least 30% of the thickness between the surfaces of the substrate at an initial thickness between the surfaces. 47. The method of claim 46, wherein the mask comprises a quartz plate having a chrome pattern. 47. The method of claim 46, wherein the corrosion material is hydrofluoric acid. 47. The method of claim 46, further comprising removing the mask prior to exposing at least a portion of the substrate to the corrosive agent. CLAIMS What is claimed is: 1. A method of producing a product having biological samples for analysis,
Providing a substrate comprising silicon and having a first surface and a second opposing surface;
Applying a pattern to one of said surfaces;
Etching a plurality of wells of the first surface;
Removing material from the second surface to form at least one through-hole from a well corresponding to the plurality of wells; And
Forming a surface roughness on at least one of the surfaces, wherein the surface roughness comprises an arithmetic mean roughness (Ra) of less than or equal to 5 nanometers, a maximum valley depth of roughness (Rv) of less than or equal to 15 nanometers, Or a peak roughness (Rt) for a maximum trench less than or equal to 24 nanometers. 56. The method of claim 55, further comprising at least partially filling at least one of the at least one through hole with a liquid solution comprising a nucleotide sequence and a fluorescent dye. A method for producing a plurality of biological samples, the method comprising:
Providing the product of claim 1;
Carrier, wherein the carrier comprises:
A first cover including a lower surface;
A second cover including an upper surface; And
An aperture sized to receive the article,
Wherein the insertion tool comprises:
U shaped body;
And a pair of arms configured to slideably engage the carrier,
Attaching the product within the U-shaped body; And
And sliding the insertion tool in an amount sufficient to position the article between the covers. 60. The method of claim 57,
Providing a wiper blade configured to contact at least one of said surfaces of said article;
Placing a quantity of liquid in one of said surfaces of said product, said liquid comprising a biological sample to deposit into said reaction sites of said product;
Further comprising: depositing the liquid on at least some of the plurality of reaction seats while sliding the insertion tool by wiping the wiper blade across at least one of the surfaces. 60. The method of claim 57, further comprising removing the insertion tool from the article and the carrier. 58. The method of claim 57, further comprising affixing the insert into the U-shaped body after attaching the insert to the carrier. 58. The method of claim 57, further comprising attaching the insertion tool and the article to the carrier together. 60. The method of claim 57, wherein at least 99% of the liquid sample is received by at least some of the reaction sites. 58. The method of claim 57, wherein at least 99% of the liquid sample is accommodated by less than 95% of the reaction sites.

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